Cell death is a fundamental aspect of animal development. A considerable proportion of the cells that are generated die during the normal development of both vertebrates (Glucksmann, Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1951)) and invertebrates (Truman and Schwartz, Ann. Rev. Neurosci. 7:171-188 (1984)). Cell death plays a role in morphogenesis (e.g., of the eye, secondary palate, heart, nervous system and limbs in vertebrate embryos), metamorphosis (e.g., in moths and other insects), and tissue homeostasis (e.g., of epithelial linings and the thymus), as well as in neuron selection during the establishment of synaptic connections and in sexual dimorphism (reviewed by Ellis et al., Ann. Rev. Cell Biol. 7:663-698 (1991)). Cell death which occurs as a part of normal development will be referred to herein as physiological cell death.
Besides physiological cell death, cell death may occur as a pathological manifestation of disease, in which case it will be referred to herein as pathological cell death (see review by Trump and Mergner (1974), in: The Inflammatory Process, vol. 1, 2nd ed. (eds. Zweifach et al.), Academic Press, New York, pp. 115-257). Cell death can result from a variety of injuries to the cell, including toxins, ischemia (lack of blood supply), hypoxia (lack of oxygen) and infectious agents, as well as from genetic mutations. The major clinical aspects of most degenerative diseases are a consequence of cell death. For example, Huntingtons's disease, Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis are marked by degeneration of neurons, while Duchenne muscular dystrophy is characterized by muscle degeneration. In addition, some cancers are thought to be caused by a defect in cell death processes. Thus, understanding and preventing cell death can be viewed as one of the major goals of biomedical research.
The simple and invariant anatomy and development of the nematode Caenorhabditis elegans have made it an attractive system for the study of cell death. Because C. elegans is small, cellularly simple and transparent, Nomarski differential interference microscopy can be used to observe individual cells throughout development. As a result, the complete cell lineage of C. elegans, from zygote to adult, has been elucidated (Sulston and Horvitz, Dev. Biol. 82:110-156 (1977); Kimble and Hirsh, Dev. Biol. 70:396-417 (1979); Sulston et al., Dev. Biol. 100:64-119 (1983)).
Cell death is an important component of the development of C. elegans: during the development of the adult hermaphrodite, the generation of 816 nongonadal cells is accompanied by the generation and subsequent deaths of an additional 131 cells. Cell death appears to be an integral part of the differentiation of a variety of tissues. The pattern of cell deaths is essentially invariant among different animals, i.e, the same set of cells die at the same developmental time. In addition, a vast majority of cell deaths in C. elegans does not appear to be initiated by interaction with surrounding cells or diffusible factors.
Genetic analysis has identified many genes that affect programmed cell death in C. elegans (reviewed by Ellis et al. (1991) supra). The activities of two genes, ced-3 and ced-4, seem to be required for the onset of almost all C. elegans programmed cell deaths (Ellis and Horvitz, Cell 44:817-829 (1986)). Mutations in ced-3 and ced-4 block essentially all programmed cell deaths. In ced-3 and ced-4 mutants, cells that normally undergo programmed cell death instead survive, differentiate and even function (Ellis and Horvitz (1986) supra; Avery and Horvitz, Cell 51:1071-1078 (1987); White et al., Phil. Trans. R. Soc. Lond. B. 331:263-172 (1991)). Genetic analyses indicate that ced-3 and ced-4 genes most likely act within dying cells; this suggests that of these genes are expressed within dying cells and either encode cytotoxic molecules or control the activities of cytotoxic molecules (Yuan and Horvitz, Dev. Biol. 138:33-41 (1990)).
Relatively little is known about the mechanism of cell death. Initiation of cell death occurs in response to a variety of signals. External injuries and cytotoxic agents cause cells to die. Endocrine signals trigger cell death during insect metamorphosis, thymocyte death and regression of the prostate in the male rat after castration. Lack of neuronal growth factors is suspected to be the cause of certain neuronal cell deaths during vertebrate development and may also be the cause of cell deaths in certain neurodegenerative diseases. A specific protein, Mullerian inhibiting substance, is responsible for the regression of the Mullerian duct during the development of male mammals. In addition, genetically programmed cell deaths which occur apparently autonomously of cell-cell interaction or diffusible factors are observed in C. elegans and other invertebrates. (Truman and Schwartz, Neuro. Comm. 1:66-72 (1982); Cohen and Duke, J. Immunol. 132:38-42 (1984); Isaacs, Prostate 5:545-557 (1984); Martin et al., J. Cell. Biol. 106:829-844 (1988); Oppenheim and Prevette, Neurosci. Abstr. 14:368 (1988); Beal et al., Nature 321:168-171 (1986); Birkmayor and Hornykiewicz, Advances in Parkinsonism, Fifth International Symposium on Parkinson's Disease, Vienna, Roche, Basle, 1976; Lagsto et al., Science 219:979-980 (1983); Rossor, Lancet 2:1200-1204 (1982); Biel et al., Science 229:289-291 (1985); Cosi et al., in: Advances in Experimental Medicine and Biology, vol. 209, Plenum Press, New York, 1987; Bonilla et al., Cell 54:447-452 (1988); Picard and Josso, Biomedicine 25:147-150 (1976)).
Cell deaths also vary morphologically. Two major categories of cell deaths have been established based on morphological features (Kerr et al., Br. J. Cancer 26:239-257 (1972)). The first type of cell death, called necrosis, is characterized by cellular swelling, rupture of plasma and internal membranes, and eventual leakage of cellular contents into the extracellular space. The second, called apoptosis, involves progressive condensation of cytoplasm and nuclear chromatin and eventual fragmentation of cellular membranes into `apoptotic bodies`, which are usually digested by macrophages or adjacent epithelial cells. Necrosis is most often a manifestation of certain pathological conditions, e.g., injury by complement (Hawkins et al., Am. J. Pathol. 68:255-288 (1972)), hypoxia (Jennings et al., Am. J. Pathol. 81:179-198 (1975)), or exposure to a variety of toxins (McLean et al., Int. Rev. Exp. Pathol. 4:127-157 (1965)). In contrast, apoptosis is usually associated with physiological conditions, e.g., embryogenesis (Bellaris, J. Anat. 95:54-60 (1961); Saunders, Science 154:604-612 (1966)) and metamorphosis (Truman, Ann. Rev. Neurosci. 7:171-188 (1984). Interestingly, morphological features of physiological cell death in C. elegans resemble, in general, those of apoptosis in vertebrates (Ellis et al., Ann. Rev. Cell Biol. 7:663-698 (1991)). However, deviations from the standard descriptions of necrosis and apoptosis are often observed. It is uncertain whether this morphological classification reflects real differences in underlying mechanisms of cell death.
Although the initiation and morphology of cell death vary, there is evidence which suggests that most physiological and some pathological cell deaths may share a common feature involving the activation of cell death genes. The existence of a genetic cell death program in a variety of organisms is suggested by the observation that protein and RNA synthesis inhibitors can prevent or delay a variety of cell deaths (insect metamorphosis, prostate regression, vertebrate neuronal cell death and thymocyte cell death) (Lockshin, J. Insect Physiol. 15:1505-1516 (1969); Stanisic et al., Invest. Urol. 16:19-22 (1978); Martin et al. (1988) supra; oppenheim and Prevette (1988) supra; Cohen and Duke (1984) supra). New RNA and protein species have been found after the initiation of cell death in the rat prostate after castration (Buttyan et al., Molecular Endocrinology 2:650-657 (1988); Lee et al., Prostate 7:171-185 (1985)). Thus, a better understanding of the mechanisms of cell death would have wide biological application and provide a basis for altering or controlling the process.